Tuberculosis vaccine

ABSTRACT

The present invention relates to an isolated microorganism belonging to the genus  Mycobacterium , characterized in that it comprises inactivating the gene Rv0757 that confers a PhoP− phenotype and inactivating a second gene that prevents the production of DIM (DIM-phenotype). Additionally, the present invention comprises the use of said microorganism for producing a vaccine for immunizing against or preventing tuberculosis.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/294,199, filed Jan. 11, 2010, now U.S. Pat. No. 8,287,866, which is aU.S. National Phase Entry of International Application No.PCT/ES20007/070051, filed Mar. 14, 2007, which claims priority toSpanish Patent Application no. P 200600761, filed Mar. 24, 2006, theentire disclosures of which are incorporated by reference herein.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to an isolated microorganism belonging tothe Mycobacterium genus, characterised in that it comprises theinactivation of the Rv0757 gene that confers a PhoP− phenotype and theinactivation of a second gene that prevents DIM production (DIM−phenotype). Additionally, the present invention comprises the use ofsaid microorganism for the preparation of a vaccine for the immunizationor prevention of tuberculosis.

The use of vaccines to prevent tuberculosis in humans has proved to be atremendous challenge for almost a century now. BCG, derived from M.bovis, is currently the only tuberculosis vaccine in use and is the mostwidely used vaccine in the world. The development and generalisedadministration of the BCG vaccine since the beginning, of the 1920srepresented a significant advance, with the prospect of being able toeradicate tuberculosis from the world. However, these initial promiseswere not achieved and, from the results of a large number of efficacytrials, it is clear that the BCG vaccine in its current form is oflimited use in controlling the disease, particularly in respiratoryforms in adults in third world areas where the disease is endemic. Fine,P. E. Variation in protection by BCG: implications of and forheterologous immunity. Lancet 1995, 346(8986), 1339-1345. With moreknowledge of the virulence of M. tuberculosis and immune response modelsthat lead to the generation of protective immunity, it is possible todevelop better vaccines than BCG. The observation that higher protectionlevels are achieved when the host is vaccinated with BCG suggests thatviability and persistence are fundamental properties required for thesuccess of a tuberculosis vaccine. In the present invention, we use a M.tuberculosis strain with the inactivated Rv0757 (phoP) gene and a secondindependent mutation of phoP, which prevents DIM synthesis, as aprototype single dose live vaccine, and we show that, as well as beingmore attenuated than BCG in immunocompromised SCID mice, it providedprotection levels comparable to those conferred by BCG in mice andhigher protection than BCG in guinea pigs.

The phoP gene, together with phoR, forms part of a two-component systemthat shows a high degree of similarity to other two-component systemsthat control the transcription of key virulence genes in intracellularpathogens. It also controls the expression of many other genes that arenot directly involved in virulence. Groisman, E. A. The pleiotropictwo-component regulatory system PhoP-PhoQ. J Bacteriol 2001, 183(6),1835-1842. The elimination of virulence genes does not seem to be, perse, the only method for the attenuation of M. tuberculosis. It was shownthat a pantothenate auxotrophic mutant of M. tuberculosis, which isincapable of de novo synthesis of pantothenic acid, persisted in SCIDmice, without managing to cause the disease. Sambandamurthy, V. K.,Wang, X., Chen, B. et al. A pantothenate auxotroph of Mycobacteriumtuberculosis is highly attenuated and protects mice againsttuberculosis. Nat Med 2002, 8(10), 1171-1174. Individual leucineauxotrophs are also strongly attenuated and incapable of replication invivo in SCID mice. Hondalus, M. K., Bardarov, S., Russell, R., Chan, J.,Jacobs, W. R., Jr. & Bloom, B. R. Attenuation of and protection inducedby a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000,68(5), 2888-2898. Therefore, the principle that vaccine strains based onM. tuberculosis can be successfully attenuated whilst retaining genesthat are suppressed in M. bovis BCG is now generally accepted.

In the past, research into more effective vaccines than BCG was based onthe notion that loss of virulence with BCG was in itself a factor thatcontributed to its lack of complete protective efficacy. Behr, M. A.,Wilson, M. A., Gill, W. P. et al. Comparative genomics of BCG vaccinesby whole-genome DNA microarray. Science 1999, 284(5419), 1520-1523. Itwas therefore reasoned that new attenuated mutants of M. tuberculosis,with less virulence, could be more effective as vaccines. However, arecent study has shown that natural infection with M. tuberculosis andvaccination with BCG do not differ in their capacity to bring aboutprotective immunity against tuberculosis. Sampson, S. L., Dascher, C.C., Sambandamurthy, V. K. et al. Protection elicited by a double leucineand pantothenate auxotroph of Mycobacterium tuberculosis in guinea pigs.Infect Immun 2004, 72(5), 3031-3037. This raises questions as to whetheror not it is possible to improve BCG by rational attenuation of M.tuberculosis. Within this context, the observation that the mutant M.tuberculosis strain of the present invention with the combination of 2independent mutations 1.—in synthesis of the PhoP protein and 2.—in DIMsynthesis is more attenuated than BCG in the SCID mouse model, even whenapplied at a dose 10 times higher than those of BCG, and the greaterdegree of protection than BCG in the guinea pig model is particularlysurprising and significant.

BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the invention relates to an isolated microorganismbelonging to the Mycobacterium genus, characterised in that it comprisesthe inactivation of the Rv 0757 (phoP) gene and the inactivation of asecond gene that prevents DIM (phthiocerol dimycocerosates) production.Hereinafter this isolated microorganism will be referred to asmicroorganism of the present invention.

A second aspect of the present invention relates to an isolatedmicroorganism belonging to the Mycobacterium genus, characterised inthat it comprises inactivating the Rv 0757 (phoP) gene and a secondindependent mutation of phoP that prevents DIM production. In apreferred aspect of the present invention, said second mutation is inthe Rv2930 (fadD26) gene, consisting of the deletion of the fadD26 gene,which is essential for DIM synthesis.

A third aspect of the present invention relates to the use of theisolated microorganism of the present invention to prepare a vaccine forthe prevention of tuberculosis in animals and still more preferably forthe prevention of tuberculosis in humans, as well as other uses thattuberculosis vaccines currently have in the treatment of diseases inhumans such as bladder cancer.

Hereinafter in the context of the present invention the “M. tuberculosisSO2 strain” will be used to refer to the isolated microorganism of theM. tuberculosis strain that has been inactivated by the Rv0757 geneconstructed from the M. tuberculosis MT103 clinical strain by insertionof a kanamycin resistance marker at the BclI site of the Rv0757 gene ofM. tuberculosis using homologous recombination according to the methoddescribed by Pelicic et al (1997) (Efficient allelic exchange andtransposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad SciUSA 94: 10955-10960) and which additionally comprises the inactivationof a second gene that prevents DIM (phthiocerol dimycocerosates)production. Therefore, said strain of the invention presents twoindependent mutations in live attenuated vaccines derived from M.tuberculosis, the independent phoP mutation not affecting the propertiesof the vaccine derived from the inactivation of said gene. Example 9describes how to construct an isolated microorganism of theMycobacterium genus with the independent double mutation that providesthe same phenotype as described for the M. tuberculosis SO2 strain.

Hereinafter in the context of the present invention vaccine will be usedto refer to those drugs whose administration produces defences againstthe disease to be prevented.

Hereinafter in the context of the present invention BCG will be used torefer to the current vaccine that has been in use against tuberculosissince 1921. It is a live attenuated vaccine derived from a M. bovisstrain that lost its virulence after being subcultured in the laboratoryand which we now know has more than one hundred deleted genes. Behr, M.A. BCG—different strains, different vaccines? Lancet Infect Dis 2002,2(2), 86-92.

Hereinafter in the context of the present invention H37Rv will be usedto refer to a pathogenic M. tuberculosis strain that has been sequenced,Cole et al. referring to these genes as Rv (Ref Cole et al 1998Deciphering the biology of Mycobacterium tuberculosis from the completegenome sequence. Nature 393: 537-544).

Hereinafter in the context of the present invention MT103 will be usedto refer to a M. tuberculosis clinical isolate. Camacho et al. 1999Identification of a virulence gene cluster of Mycobacterium tuberculosisby signature-tagged transposon mutagenesis. Mol Microbiol 34: 257-267.

Hereinafter in the context of the present invention DIM− strain will beused to refer to the strain of the M. tuberculosis complex that is notcapable of synthesizing phthiocerol dimycocerosates, which are importantlipids related to the pathogenicity of M. tuberculosis. The 1A29 strainis used in FIG. 11, which consists of the MT103 strain with the Rv2930(fadD26) gene inactivated by the transposon 1096 described in Camacho etal.

Hereinafter in the context of the present invention SO2+ pSO5 will beused to refer to the M. tuberculosis SO2 strain in which the mutation inRv0757 is complemented by the Rv0757 gene by transformation of areplicative plasmid with the mycobacterial phoP gene, but it is notcapable of complementing DIM synthesis, its phenotype being phoP+ DIM−.

Hereinafter in the context of the present invention M. tuberculosisphoP− will be used to refer to the M. tuberculosis strain that has beeninactivated by the Rv0757 gene by deletion between the EcoRV-BspEIsites, its phenotype being phoP− DIM+.

Hereinafter in the context of the present invention Rv2930 (fadD26) willbe used to refer to the gene that is at the beginning of the operon thatis responsible for the synthesis of phthiocerol dimycocerosates (Camachoet al.) and the elimination of this gene in M. tuberculosis confers astable DIM− phenotype.

Other objects, advantages and novel features of the present inventionwill become apparent from the following detailed description of theinvention when considered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot image of extracellular protein extracts.

FIGS. 2 a and 2 b shows attenuation of the SO2 strain of the presentinvention in SCID mice

FIG. 3 shows cellular immune responses in mice vaccinated with the SO2strain of the present invention and BCG.

FIGS. 4 a and 4 b shows the protective efficacy of SO2 of the presentinvention compared to BCG in vaccinated Balb/c mice.

FIGS. 5 a and 5 b shows the protective efficacy in guinea pigsvaccinated with the SO2 strain of the present invention and BCG againstlow doses of M. tuberculosis H37Rv.

FIGS. 6 a-6 d shows the protective efficacy in guinea pigs vaccinatedwith the SO2 strain of the present invention and BCG against infectionwith high doses of M. tuberculosis H37Rv.

FIG. 6 c depicts low resolution (×30) images of representative sectionsof lung lobes taken from guinea pigs of each of the treatment groups.The bar represents 1 mm.

FIGS. 7 a and 7 b show the attenuation of intravenous infection with SO2of the present invention in BalbC mice is not restored by complementingwith phoP.

FIGS. 8 a and 8 b show that the SO2 strain does not produce DIM and DIMsynthesis.

FIG. 9 shows the construction of plasmids for the inactivation of thefadD26 gene.

FIG. 10 shows the construction of plasmids for the inactivation of thephoP gene.

FIG. 11 shows the study of attenuation in mice.

FIG. 12 shows the weight curve of guinea pigs at 50 times the vaccinedose of SO2.

FIG. 13 shows the survival rate of vaccinated guinea pigs afterinfection with M. tuberculosis.

FIG. 14 a ₁-14 b ₃ shows the weight curve of the studied guinea pigsafter 400 days.

DETAILED DESCRIPTION

The Western blot analysis of FIG. 1 is a blot image of extracellularprotein extracts of MT103, the SO2 strain of the present invention andBCG Pasteur, using polyclonal antibodies raised against PhoP and ESAT-6.The MT103 strain has a ESAT6+ and phoP+ phenotype, the SO2 strain has aPhoP− and ESAT6+ phenotype and the BCG vaccine strain is PhoP+ andESAT6−.

Attenuation of the SO2 strain of the present invention in SCID mice isillustrated by FIGS. 2 a and 2 b. The survival rate curve for aerosolinfected SCID mice (n=10), with 20 CFUs of SO2, SO2 complemented withpSO5 (SO2+pSO5) and MT103. The mean number of days of survival was morethan 245 days (SO2), 62.1±5.88 (SO2+pSO5) and 36.7±0.67 (MT103). Themice infected by aerosol with the SO2 strain survived for the 245 daysof the experiment whereas those infected by MT103 and the SO2 straincomplemented with phoP died before day 62 as shown in FIG. 2 a. FIG. 2 bdepicts the survival rate curves for SCID mice (n=7) infected byintravenous injection with 5.4×10⁶ CFUs of SO2 and 2×10⁵ CFUs of BCGPasteur. This shows that the attenuation level of the SO2 strain isgreater than that of BCG, the tuberculosis vaccine currently used inhumans.

The cellular immune responses in mice vaccinated with the SO2 strain ofthe present invention and BCG was also studied. Balb/c mice werevaccinated by subcutaneous injection with 8×10³ CFU of BCG (Phipps) or2.5×10³ CFU of the SO2 strain of the present invention. The results areshown in FIG. 3 as a percentage of the total CD4+/CD8+ (populations inthe spleen at intervals of time after vaccination and as a percentage ofcells that express IFNγ of the total CD4+/CD8+ population afterstimulation with complete M. tuberculosis antigen. *denotesstatistically significant differences between the groups at the givenpoints of time (p<0.005). The cellular immunity results show that thenumber of CD4+ lymphocytes in the animals vaccinated with the SO2 strainis greater on days 14, 30, 45 and 60 and the production of specific IFNγagainst the M. tuberculosis antigens is significant on days 45 and 60 inrelation to the mice vaccinated with BCG. The number of CD8+ lymphocytesin the animals vaccinated with the SO2 strain is greater on days 45 and60 and the production of specific IFNγ against the M. tuberculosisantigens is significant on day 14 in relation to the mice vaccinatedwith BCG.

The protective efficacy of SO2 of the present invention compared to BCGin vaccinated Balb/c mice was measured by the number of CFUs recoveredfrom lungs (FIG. 4 a) and spleens (FIG. 4 b) of Balb/c mice vaccinatedwith the SO2 strain of the present invention and BCG, intravenouslyinfected with M. tuberculosis H37Rv. The reduction of CFUs in the lungand spleen of mice vaccinated with SO2 is similar to that obtained inthose vaccinated with BCG and it shows significant protection inrelation to the non-vaccinated mice.

The protective efficacy in guinea pigs vaccinated with the SO2 strain ofthe present invention and BCG against low doses of M. tuberculosis H37Rvwas evaluated. Mean numbers of log₁₀ CFU/ml in the lungs (FIG. 5 a) andspleens (FIG. 5 b) of vaccinated guinea pigs and control guinea pigsinjected with saline solution infected with low doses of M. tuberculosisH37Rv. The data represent the mean CFUs of all the animals (n=6)sacrificed after 4 weeks. Error bars indicate the standard deviation.The reduction in CFUs in the lungs and spleen of guinea pigs infectedwith M. tuberculosis at low doses and vaccinated with SO2 is similar tothat obtained in those vaccinated with BCG and is significant inrelation to the unvaccinated mice.

Given that the protection experiments in mice and in guinea pigsinfected at low doses showed a clear protection in the mice vaccinatedwith SO2 and BCG but no differences between BCG and SO2, a guinea pigmodel with infection at high doses was used. Survival rate curve forguinea pigs after aerosol infection with M. tuberculosis H37Rv isillustrated in FIG. 6 a while the extent of lung disease and spread ofinfection, measured by total lung consolidation, is shown in FIG. 6 b.The values of each individual animal sacrificed at the human end pointare marked with an “x”. The broken line indicates the mean value inpercentage of the group (# in SO2 corresponds to two animals). FIG. 6 cis a low resolution (×30) of images of representative sections of lunglobes taken from guinea pigs of each of the treatment groups. The barrepresents 1 mm. In FIG. 6 d, mean CFU counts in the spleen and lungs ofvaccinated and unvaccinated guinea pigs are compared. This experimentshows that with the guinea pig model at high doses of infection with M.tuberculosis, the guinea pigs vaccinated with SO2 survived significantlylonger than with BCG vaccination and they also produced fewer lunglesions and had a lower number of CFUs in the spleen and lungs inrelation to the current BCG vaccine.

Intravenous infection in Balb/C mice with 105 CFUs of the SO2(phoP−DIM−) strain compared to the wild-type MT103 strain and the straincomplemented with phoP (SO2+pSO5) was studied. The attenuation ofintravenous infection with SO2 of the present invention in BalbC mice isnot restored by complementing with phoP. A reduction in colonies (CFUs)was observed in both spleen (FIG. 7 a spleen) and lung (FIG. 7 b lung),measured after 3 and 6 weeks. The levels of CFUs of the wild-type strainwere not restored in the complemented strain. These experiments inimmunocompetent mice suggest that the surprising attenuation could bedue to a second additional mutation that is not restored by phoPcomplementation.

Analysis of lipids from different strains of M. tuberculosis by thinlayer chromatography shows the SO2 strain of the present invention doesnot produce DIM and DIM synthesis is independent of the phoP mutation.DIM production can be observed in FIG. 8 a with the MT103 strain,whereas DIM is not produced with the SO2 strain and complementation withthe phoP (SO2 pSO5) gene. This shows that with SO2 the absence of DIM isindependent of phoP. FIG. 8 b shows the MT103 strain and the MT103strain inactivating only the phoP (MT103 ΔphoP::hyg) gene and both arecapable of synthesising DIM, which confirms that DIM production isindependent of the phoP mutation.

Construction of plasmids for the inactivation of the fadD26 gene isshown in FIG. 9. Construction of plasmids for the inactivation of thephoP gene is shown in FIG. 10.

Survival rate curve for Balb/C mice intratracheally inoculated to studythe attenuation of the different strains of M. tuberculosis is shown inFIG. 11. H37Rv and MT103 correspond to strains of M. tuberculosiswithout mutations and all the mice died before the 10^(th) week. Withthe M. tuberculosis DIM− (1A29) strain, 50% of the mice had survivedafter 20 weeks. All the animals inoculated with SO2 (phoP− and DIM−mutant) survived for the 20 weeks of the experiment.

To show that SO2 is not toxic six guinea pigs were inoculated with 50times the vaccine dose. Their survival rate and weight curve wasstudied. The survival rate was 100% after the 6-month duration of theexperiment. FIG. 12 shows the observed weight gain in all the animalsover the 6 months, showing the non-toxicity of the SO2 strain (Y=weightin grams and week of infection. X=time in weeks).

The survival rate of vaccinated guinea pigs after infection with M.tuberculosis was studied (FIG. 13). The protection study in guinea pigstracked the survival rate of guinea pigs, after 300 days. The survivalrate curve was measure for unvaccinated guinea pigs (saline) and thosevaccinated with the current BCG vaccine, with a M. tuberculosis phoP−strain or with SO2 (phoP− and DIM− mutant). After subcutaneousvaccination, the animals were infected with a virulent strain of M.tuberculosis (H37Rv) at a high dose to study the survival rate. After 60days the 6 guinea pigs that had not been vaccinated (saline) had died,whilst the groups vaccinated with SO2, phoP− and BCG had survived. After300 days of infection 3 guinea pigs vaccinated with BCG and phoP− haddied, compared to only one of the group vaccinated with SO2, whichindicates that the protection of the phoP mutant is similar to that ofthe current vaccine BCG, whereas vaccination with SO2, the phoP− andDIM− double mutant, protects better in the guinea pig model.

FIG. 14 a ₁-14 a ₃ show the survival after 400 days of the guinea pigstracked in FIG. 13. The 6 unvaccinated guinea pigs had died after 60days. After 400 days of infection 3 guinea pigs from the groupvaccinated with SO2 (FIG. 14 a) survived, whereas just 1 guinea pigvaccinated with BCG (FIG. 14 a and FIG. 14 b) and phoP− (FIG. 14 b) hadsurvived, indicating again that the protection of the phoP mutant issimilar to that of BCG, whilst vaccination with SO2, the phoP− and DIMdouble mutant, protects better after the 400 days of the experiment.

One aspect of the present invention relates to an isolated microorganismbelonging to the Mycobacterium genus, characterised in that it comprisesthe inactivation of the Rv0757 gene that confers a PhoP− phenotype andthe inactivation of a second gene that prevents the DIM production (DIM−phenotype). Additionally, the present invention comprises the use ofsaid microorganism for the preparation of a vaccine for the preventionof tuberculosis, and the vaccine per se.

Throughout the present invention, it is shown how isolated phoP− DIM−strains of the genus Mycobacterium present characteristics that makethem particularly suitable for use as vaccines, due to both the level ofattenuation, that they acquire and the level of protection that theyconfer.

In order to demonstrate the attenuation immunodepressed SCID mice wereinoculated by aerosol with the SO2 (phoP− DIM−) strain. Said micesurvive (FIG. 2 a) significantly longer than mice infected by thewild-type strain. Additionally, this attenuation is complemented withphoP in the SO2+pSO5 (phoP+ DIM−) strain (FIG. 8 a).

Moreover, when attenuation studies are carried out in immunocompetentBalb/C mice by intravenous injection (FIG. 7), there is a clearattenuation of SO2 in relation to the wild-type MT103 strain, butsurprisingly this attenuation is not complemented with phoP, as theSO2+PSO5 (phoP+ DIM−) strain is as virulent for the immunocompetentmouse as the wild-type strain. Survival studies in Balb/C mice comparingthe SO2 (phoP− and DIM−) strain with just a DIM− strain show asurprisingly higher survival rate for SO2 (FIG. 11).

Comparative survival studies of SO2 and BCG in intravenously infectedSCID mice show that the level of attenuation of the SO2 strain is higherthan that of BCG, the vaccine that is currently used in humans againsttuberculosis (FIG. 2 b). Toxicity studies in guinea pigs with 50 timesthe dose of vaccine used in quality control for batches of BCG vaccineshow that over the 6 months of the study the guinea pigs gain weight anddo not present macroscopically or microscopically visible histologicallesions that are compatible with tuberculosis, thereby confirming theattenuation and non-toxicity of SO2 (FIG. 12). This surprisingattenuation and lack of toxicity is due to the PhoP− DIM− phenotype andalso these mutations remain sensitive to antituberculosis drugs, whichwould allow a conventional treatment.

It is shown herein that in vaccination experiments carried out in Balb/cmice, the levels of protection conferred by the M. tuberculosis SO2strain of the present invention and BCG were similar in both the lungsand the spleen up to four weeks after infection. If we compare therelative proportions of CD4+ and CD8+ cells from the spleens ofvaccinated mice, in the mice vaccinated with the SO2 strain of thepresent invention a higher percentage of both CD4+ and CD8+ cells wasfound compared to the mice vaccinated with BCG. Furthermore, when thesecells were stimulated with antigens derived from culture filtrate, asignificantly higher percentage of CD4+/IFN-γ+ was measured in the micevaccinated with the SO2 strain of the present invention 45 and 60 daysafter vaccination. Although it is not significant at each point of time,a similar tendency was measured for CD8+/IFN-γ+ in the mice vaccinatedwith the SO2 strain of the present invention. The data suggest thatvaccination with the SO2 strain of the present invention results inbetter T cell activation compared to vaccination with BCG, measured byIFNγ synthesis. Given that protective immunity against M. tuberculosisgenerally depends on the generation of a TH₁-type cellular immuneresponse characterised by the secretion of IFN-γ from the specific Tcells of the antigen, it can be concluded that the relatively highlevels of T cell activation induced by the SO2 strain of the presentinvention contributes to its capacity to confer a strong protectiveresponse.

Additionally, by using different systems and test models and a varietyof conditions, we have managed to show the mouse model's relativecapacity for studying the differences in protection of BCG compared toSO2. It was shown that the two vaccines, SO2 (phoP− DIM−) and BCG,confer protection in the mouse model.

A strategy was undertaken to compare the vaccines in a more significantand gradually more demanding trial with guinea pigs. This systematicapproach to the comparison of vaccines could represent a useful startingpoint for identifying the best candidate vaccines for which furthertrials should be conducted. It is generally accepted that guinea pigsare more susceptible to infection by tuberculosis and could therefore bea more significant model for this disease. Baldwin, S. L., D'Souza, C.,Roberts, A. D. et al. Evaluation of new vaccines in the mouse and guineapig model of tuberculosis. Infect Immun 1998, 66(6), 2951-2959. Theadvantage of the guinea pig compared to mice is that the pathology ofthe disease is similar to that observed in tuberculosis in humans and itis therefore an appropriate model for testing the efficacy of a vaccine.In a recent aerosol vaccine study with a double pantothenate and leucineauxotrophic mutant of M. tuberculosis, protection levels equivalent toM. bovis BCG were obtained in the lungs and spleen of vaccinated guineapigs, with limited spreading of the infection to the spleen induced byboth vaccines, five weeks after aerosol application of M. tuberculosis.Sampson, S. L., Dascher, C. C., Sambandamurthy, V. K. et al. Protectionelicited by a double leucine and pantothenate auxotroph of Mycobacteriumtuberculosis in guinea pigs. Infect Immun 2004, 72(5), 3031-3037. Inanother study that used recombinant BCG that expressed ESAT-6, higherprotection levels than M. bovis BCG were only observed in the spleenPym, A. S., Brodin, P., Majlessi, L. et al. Recombinant BCG exportingESAT-6 confers enhanced protection against tuberculosis. Nat Med 2003,9(5), 533-539., suggesting that the improved protection is limited toits ability to prevent the infection from spreading from the lung.

To perform the present infection, guinea pigs were inoculated with a lowdose of M. tuberculosis H37Rv, and the protection levels conferred byvaccination with the SO2 strain of the present invention and BCG weresimilar in both the lungs and the spleen up to 4 weeks after infection.Both vaccines provided extremely efficient protection, reducing the CFUsin the lungs and spleen by approximately 2 log. compared to the controlgroups, which received saline solution. However, there was nostatistically significant difference between the two vaccine groups. Insuch a short period after infection, we can assume that it would bedifficult to prove the greater efficacy of a new vaccine in relation toBCG. This is due to the fact that, at present, the CFUs (colony-formingunits) of the organs of animals vaccinated with BCG are so low that thetest does not have the differentiating capacity to show a significantadditional reduction in CFUs. In other survival studies with guinea pigsit has been shown that, although vaccination with BCG provides astatistically significant protection compared to unvaccinated controls(or vaccinated with ineffective vaccines), this protection is onlypartial even against challenge with low doses of M. tuberculosis. Instudies with an application of low doses conducted over 60 to 80 weeksafter infection, some controls with BCG did not protect any of theguinea pigs, Horwitz, M. A. & Harth, G. A new vaccine againsttuberculosis affords greater survival after challenge than the currentvaccine in the guinea pig model of pulmonary tuberculosis, Infect Immun2003, 71(4), 1672-1679, whilst others protected a low percentage(between 20 and 30%) of the animals, Brandt, L., Skeiky, Y. A.,Alderson, M. R. et al. The protective effect of the Mycobacterium BovisBCG vaccine is increased by coadministration with the Mycobacteriumtuberculosis 72-kilodalton fusion polyprotein Mtb72F in M.tuberculosis-infected guinea pigs. Infect Immun 2004, 72(11), 6622-6632and Wiegeshaus, E. H., McMurray, D. N., Grover, A. A., Harding, G. E. &Smith, D. W. Host-parasite relationships in experimental airbornetuberculosis. 3. Relevance of microbial enumeration to acquiredresistance in guinea pigs. Am Rev Respir Dis 1970, 102(3), 422-429.Application at a high dose, on the other hand, may result in more severedisease than that normally used to evaluate the protective efficacy ofTB vaccines.

For the present invention we used aerosol infection with a relativelyhigh dose of M. tuberculosis H37Rv and the study period was extended to180 days. We did this to generate a more demanding level of challengethat could show the potential protective efficacy of the SO2 strain ofthe present invention, and at the same time to facilitate a level ofdiscrimination in relation to BCG. In terms of survival, the animals ofthe group vaccinated with BCG were significantly protected in comparisonwith the unvaccinated controls, and they showed an overall protectionlevel similar to that observed in other studies, despite the relativelyhigh dose of infection used in our study. Moreover, we also found astatistically significant increase in the protective efficacy of the SO2(phoP− DIM−) strain of the present invention in comparison with BCG,measured by several indicators, including prolonged survival and thedegree of consolidation of pulmonary lesions. This less severe form ofdisease could have been directly responsible for the higher survivalrate of the animals vaccinated with the SO2 strain of the presentinvention.

The results described in the present invention show that the SO2 strainand therefore a microorganism belonging to the Mycobacterium genus(particularly from the M. tuberculosis complex) with phoP− DIM−phenotype is a more effective vaccine than BCG in accordance with anumber of criteria. It is more attenuated than BCG in SCID mice, itprovides mice with a protective immunity that is as least as good as BCGand it generates stronger cellular immune responses. Additionally, inprotection experiments conducted in guinea pigs against infection withhigh doses of H37Rv, the strain with phenotype DIM− phoP− results in a100% survival rate of guinea pigs in circumstances in which BCG onlyachieve a 33% survival rate. This protection is linked to a reduction inthe severity of the disease and the bacterial load.

In order to check whether the protection level of SO2 (phoP− DIM−) wasdue to the phoP mutation or whether it might be due to the additionalmutation in DIM, another vaccination experiment was carried out inguinea pigs with high doses of infection. Groups of 6 animals werevaccinated with BCG, with SO2 (PhoP− DIM−) and with M. tuberculosisphoP− DIM+ and 6 animals used as a control were not vaccinated. Theexperiment lasted 400 days.

In this other experiment the unvaccinated guinea pigs died before day70. After 300 days of infection 3 guineas pigs vaccinated with BCG andphoP− DIM+ had died, compared to only one in the group vaccinated withSO2, which suggests that the protection provided by the phoP− DIM+mutant is similar to the current vaccine BCG, whilst vaccination withSO2, the double phoP− and DIM− mutant, protects better in the guinea pigmodel (FIG. 13). After 400 days 3 guinea pigs in the group vaccinatedwith SO2 (FIG. 14 a) had survived, whereas only 1 guinea pig vaccinatedwith BCG (FIG. 14 a and FIG. 14 b) and phoP− DIM+ (FIG. 14 b) hadsurvived, indicating that the protection of the phoP− DIM+ mutant issimilar to BCG, whilst vaccination with SO2, the phoP− and DIM− doublemutant, protects better after the 400 days of the experiment, thesurprising effect of greater protection than BCG being attributed notonly to the phoP− mutation but to the SO2 double mutation phoP− DIM−.

Therefore, a first aspect of the present invention relates to anisolated microorganism belonging to the Mycobacterium genus,characterised in that it comprises the inactivation or deletion of:

-   -   a. the phoP gene or one or more genes that regulate the phoP        gene or that are regulated by phoP and    -   b. a second gene that prevents DIM production.

In a preferred embodiment of the invention the isolated microorganism ofthe invention is characterised in that the phoP gene is inactivatedthrough the inactivation or deletion of the Rv0757 gene.

In a more preferred embodiment of the invention the isolatedmicroorganism of the invention is characterised in that DIM productionis inactivated through the deletion or inactivation of the Rv2930(fadD26) gene.

In an even more preferred embodiment of the invention, the isolatedmicroorganism of the invention is characterised in that it comprises thedeletion or inactivation of the Rv2930 and Rv0757 genes.

In another embodiment of the invention, the isolated microorganism ofthe invention is characterised in that the species of the Mycobacteriumgenus belongs to the Mycobacterium tuberculosis complex.

A second aspect of the invention relates to the process for preparingthe isolated microorganism of the invention, which comprises:

-   -   a. The inactivation or deletion of the phoP gene or one or more        genes that regulate the phoP gene, preferably the inactivation        or deletion of the Rv0757 gene, and    -   b. The inactivation or deletion of a second gene that prevents        DIM production, preferably the deletion or inactivation of the        Rv2930 (fadD26) gene.

A third aspect of the invention relates to a vaccine (hereinafter thevaccine of the invention) to immunize an individual against the symptomscaused by tuberculosis, wherein said vaccine comprises at least oneisolated microorganism of the invention.

In a preferred embodiment of the invention, the vaccine also comprisespharmacologically acceptable excipients.

A fourth aspect of the invention relates to the process for preparing amedicine, preferably a vaccine, which comprises the incorporation of anisolated microorganism of the invention to a suitable medium foradministration in humans or animals in a therapeutically effective doseand, optionally, the addition of excipients that are pharmacologicallysuitable for the production of vaccines.

Said medicine is suitable for the treatment of bladder cancer, for thetreatment or prevention of tuberculosis, or as a vector or adjuvant,preferably to immunise an individual against the symptoms caused bytuberculosis.

A fifth aspect of the invention relates to the use of the isolatedmicroorganism of the invention to prepare the vaccine of the inventionfor the prevention and/or treatment of tuberculosis in humans oranimals.

Throughout the description and claims the word “comprise” and itsvariants do not imply the exclusion of other technical characteristics,additives, components or steps. For a person skilled in the art, otherobjects, advantages and characteristics of the invention will arisepartly out of the description and partly when the invention is put intopractice. The following examples and figures are provided by way of anon-limiting, illustrative example of the present invention.

EXAMPLES Example 1 Materials and methods

1.1.—Protein Extraction and Immunoblotting.

Polyclonal antibodies against the PhoP protein were obtained, whichreceived four doses of PhoP (0.5 mg), in weeks 0, 4, 8, 12 and 16,respectively. The anti-PhoP antibodies were detected using the ELISAtest (ZEU-Immunotec Zaragoza, Spain). Monoclonal antibodies againstESAT-6 were kindly supplied by Pym, A. S., Brodin, P., Brosch, R.,Huerre, M. & Cole, S. T. Loss of RD1 contributed to the attenuation ofthe live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacteriummicroti. Mol Microbiol 2002, 46(3), 709-717. Cell-free protein extractsof mycobacteria were prepared from early cultures in log-phase that weregrown in Middlebrook 7H9-ADC broth, following the usual methods.Sambrook, J. a. R., D W. Molecular Cloning a laboratory manual, ColdSpring Harbor Laboratory Press, New York, 2001. The M. tuberculosisprotein extracts were filtered through a Millex-GP filter with a poresize of 0.22 μm (Millipore, Bedford, Mass.). The M. tuberculosis H37Rvculture filtrate cultured for 5-6 weeks was collected and the culturefiltrate proteins were precipitated with 45% (w/v) ammonium sulphate.The Western Blot analysis was carried out according to the normalmethods. Goat anti-rabbit antibodies labelled with horseradishperoxidase (Bio-Rad Laboratories, Hercules, Calif.) were used assecondary antibodies.

1.2.—Infection of SCID Mice with M. tuberculosis.

The work with SCID mice was carried out under the supervision of theAnimal Care Committee at the “Germans Trias i Pujol” UniversityHospital, in accordance with EU laws on the protection of laboratoryanimals. SCID CB-17/Icr Ico specific pathogen free (spf) mice wereobtained from Charles River (Bagneux Cedex, France). For aerosolinfection, mice were placed in the exposure chamber of an airborneinfection apparatus (Glas-col Inc., Terre Haute, Ind., USA). Thenebuliser compartment was filled with 7 ml of a M. tuberculosissuspension to provide an approximate uptake of 20 viable bacilli withinthe lungs. Ten mice were used for each experimental group. Forintravenous infection, groups of 7 mice were infected with 200 μl of PBScontaining doses equivalent to 2×10⁵, 2×10⁴ and 2×10³ of viable BCG and5.4×10⁶, 5.4×10⁵ and 5.4×10⁴ of viable M. tuberculosis phoP strain by alateral tail vein. The significance of differences in survival timesbetween treated mice was determined using the Mantel-Haenszel test.Viable counts were performed on serial dilutions of the homogenate,plated onto Middlebrook 7H11+OADC agar and examined after 3 weeks forgrowth. For histological analysis, the tissues were fixed in bufferedformol-saline solution and embedded in paraffin. Five-μm thick sectionswere cut and stained with Ziehl-Neelsen stain.

1.3.—Determination of the Activation of Cellular Immunity in Balb/c Miceafter Subcutaneous Vaccination with SO2 of the Present Invention andBCG.

Groups of four Balb/C mice were sacrificed on days 7, 14, 21, 28, 45 and60 after subcutaneous vaccination with 8×10³ CFU of BCG (Phipps) or2.5×10³ CFU of the SO2 strain of the present invention. The spleens wereremoved and placed in 2 ml of RPMI medium and 10% foetal calf serum(GIBCO. Invitrogen Corporation) containing 0.5 mg/ml type-II collagenase(Worthington, N.J., USA), and 2 U/ml DNase (GIBCO), and incubated for 1hour at 37° C., with 5% CO2. They were then passed through a 70 μm cellsieve (Falcon, Becton Dickinson 70 μm Nylon 35-2350), crushed with asyringe plunger and rinsed with medium. The cells were centrifuged, thesupernatant was discarded and the red cells were removed with lysisbuffer. Arriaga, A. K., Orozco, E. H., Aguilar, L. D., Rook, G. A. &Hernandez Pando, R. Immunological and pathological comparative analysisbetween experimental latent tuberculous infection and progressivepulmonary tuberculosis. Clin Exp Immunol 2002, 128(2), 229-237. Aftercentrifuging and washing with RPMI medium, the cells were resuspended inFACS buffer (PBS 1×, pH 7.2, 1% BSA), and were counted. The cell surfacewas labelled by incubating 10⁶ cells with 100 μl of anti-CD4-FITC oranti-CD8-FITC monoclonal antibodies, diluted at 1:20 in PBS containing1% BSA and 0.1% sodium azide for 20 min at 4° C., and analysed using aFACScan cytometer.

The M. tuberculosis H37Rv strain was cultured in Middlebrook 7H9 medium(Difco Laboratories) supplemented with OADC (Difco Laboratories). After1 month of culture, the bacterial mass was separated and the culturefiltrate was collected. The antigens of said filtrate were precipitatedwith 45% (w/v) ammonium sulphate, washed and dissolved again in PBS. Tostimulate the cells, 1×10⁶ spleen cells were resuspended in 100 μl RPMImedium per well, and incubated with 10 μg M. tuberculosis culturefiltrate antigens, suspended in 100 μl PBS for 72 hours at 37° C. with5% CO2. The cells and the culture medium were centrifuged, thesupernatant was discarded and, after counting and checking theviability, 2.5×10⁵ cells per tube were labelled on the surface of CD4+or CD8+ cells, as has been described above. After washing, the cellswere resuspended and incubated for 20 min at 4° C. in 0.1% saponindissolved in PBS. Intracellular IFN-γ was detected by incubating thecell for 20 min at 4° C. in the dark, with 100 μl of a 1/20 dilution ofmonoclonal anti-IFN-γ labelled with phycoerithrin (PE). The cells werefixed with 100 μl of 4% paraformaldehyde diluted in PBS. The sampleswere analysed after 20 minutes using a FACScan cytometer. The isotypecontrols were Ab-FITC (1:20 dilution)+Ab-PE (1:20 dilution).

1.4.—Protective Efficacy of SO2 of the Present Invention in Balb/c Mice.

All of the animals were kept in controlled conditions in the P3 HighSecurity Laboratory of the Animal Facility at the Pasteur Institute inParis, in accordance with EU directives on the protection of laboratoryanimals. Groups of Balb/c mice (7 per group) were subcutaneouslyvaccinated at the base of the tail with 10⁷ CFUs of the SO2 strain ofthe present invention or BCG (Pasteur). Eight weeks after vaccination,all the mice were intravenously injected with 2.5×10⁵ CFU of M.tuberculosis H37Rv. Four weeks after injection, the mice weresacrificed. Viable counts were performed on serial dilutions of thehomogenate, cultured in Middlebrook 7H11+ agar OADC broth, and after 3weeks the growth of M. tuberculosis H37Rv of the SO2 strain of thepresent invention was examined on the basis of the kanamycin resistancephenotype of the latter strain.

1.5.—Protective Efficacy of SO2 of the Present Invention in Guinea Pigs.

The experimental work with guinea pigs was carried out in accordancewith UK laws on experiments on animals and was approved by a localethics committee of the Health Protection Agency, Porton Down, UK.Female Dunkin-Hartley guinea pigs were obtained from approved commercialsuppliers (UK Home Office) (David Hall, Burton-on-Trent, UK or HarlanLtd UK, Bicester, UK), and they were reared in complete isolation. Theresults presented in FIG. 6 show that the SO2 strain confers greaterprotection than BCG. The results presented in FIG. 13 and FIG. 14 showthat this surprising protection of the SO2 mutant is due to its doublephenotype DIM−/PhoP−.

1.6.—Low Dose Application.

Groups of 6 guinea pigs were subcutaneously vaccinated in the back ofthe neck with 250 μl of: 5×10⁴ CFUs of BCG Pasteur; 5×10⁴ CFUs of SO2 ofthe present invention; or with saline solution. The animals were restedfor a period of 12 weeks before aerosol challenge using a containedHenderson apparatus, as has been described above. Williams, A., Davies,A., Marsh, P. D., Chambers, M. A. & Hewinson, R. G. Comparison of theprotective efficacy of bacille calmette-Guerin vaccination againstaerosol challenge with Mycobacterium tuberculosis and Mycobacteriumbovis. Clin Infect Dis 2000, 30 Suppl 3, S299-301. Aerosols weregenerated from fine particles of M. tuberculosis H37Rv, with a meandiameter of 2 μm, (diameter range: 0.5-7 μm) using a Collison nebuliser,and applied directly to the animal's snout. The aerosol was generatedfrom a suspension in water containing 2×10⁶ CFU/ml to achieve a retainedinhaled dose that was calculated to be approximately 10-50 CFU/lung.

Four weeks after application the protection was evaluated. The animalswere sacrificed by a peritoneal overdose of sodium pentobarbital. Tissuewas aseptically removed from the spleen and lungs (the left and middlecranial lobes, right middle lobe and right caudal lobes) and placed insterile receptacles. The material was stored at −20° C. and was thenprepared to count the number of bacteria. The tissue was homogenized in10 ml (lung) or 5 ml (spleen) of sterile deionized water using arotating blade macerator system (Ystral). Viable cell counts wereperformed on serial dilutions of the homogenate, cultured in Middlebrook7H11+agar OADC, and M. tuberculosis growth was examined after 3 weeks.The data were transformed into log₁₀ for their analysis and the numbersof viable M. tuberculosis for each vaccine group were compared with thecontrol group with saline solution by Student's t-test.

1.7.—Protection Test in Guinea Pigs after Infection with a High Dose ofM. tuberculosis.

Groups of 6 guinea pigs were subcutaneously vaccinated with 5×10⁴ CFU ofSO2 of the present invention or BCG (Danish 1331) 10 weeks beforeaerosol application with M. tuberculosis. The aerosol application wasperformed as described in the previous paragraph, using a suspension of5×10⁷CFU/ml to provide the lungs with around 500 CFU. After application,the animals were kept at containment level 3 (ACDP), changes in weightwere regularly controlled and they were humanely sacrificed 180 daysafter application or at the human end point (loss of 20% of the maximumbody weight). The post-mortem sample collection and processing werecarried out as described above, except that lung consolidation wasmeasured using image analysis of sections of lung tissue fixed informalin, stained with Haematoxylin and Eosin (H+E). Animal survival wascompared using Kaplan Meier survival estimates and Log Rank distributionanalysis was used to identify the statistically significant differences.The CFU and lesion consolidation data were analysed by ANOVA, usingFisher's pairwise comparisons to compare the mean values of the groups.

Example 2 Characterisation of M. tuberculosis phoP

Evidence for the involvement of the phoP gene in global regulation ofmycobacteria genetic circuits was provided by the observation of changesin bacillus size and cording properties of growing cells harbouring theinactivated phoP gene. Given the key properties of secreted antigens asdeterminants of protection against tuberculosis, we hoped to determinewhether the pleiotropic effects of the phoP gene mutation extended toinfluence synthesis of the major immunodominant antigen: ESAT-6. Westernblot analysis was carried out on the SO2 strain, BCG and MT103, usingantibodies raised against the PhoP protein and ESAT-6. The resultsclearly showed that the PhoP protein was expressed constitutively in theM. tuberculosis MT103 and BCG strains, whilst it was completely absentin the SO2 strain of the present invention. In contrast, the levels ofexpression of ESAT-6 in the supernatant of cultures of the SO2 strainwere similar to those detected from the parental strain of MT103 and, asexpected, no ESAT-6 protein was detected in BCG.

Example 3 Survival of Mice Infected with the Strains of the PresentInvention and BCG

The survival of immunocompromised SCID mice was evaluated after aerosolinfection (approximately 20 CFU) with the MT103 strain, with SO2 andwith SO2 complemented with the phoP gene (SO2+pSO5). Perez, E., Samper,S., Bordas, Y., Guilhot, C., Gicquel, B. & Martin, C. An essential rolefor phoP in Mycobacterium tuberculosis virulence. Mol Microbiol 2001,41(1), 179-187. All of the mice infected with SO2 survived for more than245 days. In contrast, all of the SCID mice infected with MT103 orcomplemented M. tuberculosis SO2−pSO5 had died 62 days after infection,indicating a recovery of the virulence of the complemented strain (FIG.2 a).

The attenuation of the SO2 strain was also compared with BCG in SCIDmice after intravenous administration. Groups of SCID mice wereinoculated with a number of doses (2×10⁵, 2×10⁴ and 2×10³ CFU) of BCGPasteur or the SO2 strain (5.4×10⁶, 5.4×10⁵ and 5.4×10⁴ CFU) by alateral tail vein. Histological staining of the infected alveolarmacrophages of a subgroup of mice sacrificed three weeks after infectionrevealed a smaller number of alcohol-acid resistant bacilli in the lungsof mice infected with the M. tuberculosis SO2 strain, compared with BCG.All the mice inoculated with the higher doses of BCG (2×10⁵ CFU) haddied 92 days after infection (mean survival time: 89±3.5 days) (FIG. 2b). In contrast, all of the mice infected with the highest dose of SO2(5.4×10⁶ CFU) had survived after 120 days (FIG. 2 b). At the time ofdeath, the bacterial loads of the lungs of mice infected with BCG, 2×10⁵CFU, were at least 100 times higher, if compared with the mice infectedwith SO2, 5.4×10⁶ CFU.

Example 4 Quantitative CD4+ and CD8+ Responses of Vaccinated Balb/c Mice

To compare the activation of cellular immunity induced by vaccinationwith SO2 of the present invention and BCG, on days 7, 14, 30, 45 and 60after vaccination, cell suspensions were collected from the spleen ofgroups of at least four Balb/c mice subcutaneously vaccinated with theSO2 strain of the present invention and BCG Phipps, and the relativeproportions of CD4+ and CD8+ cells were determined by cytofluorometry(FIG. 3). Vaccination with SO2 induced a significantly higher number ofCD4+ cells 14 days after vaccination, compared to vaccination with BCG,and a significantly higher number of CD8+ cells after 45 days. Thesesplenocytes were stimulated with total antigens derived from M.tuberculosis culture filtrate. After 3 days, the lymphocyte populationswere analysed by flow cytometry, and specific antibodies were combinedfor the detection of CD4+/CD8+ cells and intracellular synthesis ofIFN-γ. Vaccination with SO2 induced a significantly higher proportion ofCD4+/IFN-γ+ producing cells 45 days after vaccination, compared with BCG(FIG. 3). After a certain point in time, the proportion of cells thatproduced CD8+/IFN-γ+ was always higher in the SO2 group (significantlydifferent on day 14).

Example 5 Protective Immunity Generated by SO2 of the Present Inventionin Balb/c Mice

Having proven that the SO2 strain of the present invention wasattenuated in SCID mice, we were interested to determine whether theobserved reduction in virulence would confer some kind of protectiveproperty on the mutant strain. We subcutaneously vaccinated Balb/c micewith the SO2 strain of the present invention or with BCG (Pasteur).Eight weeks after vaccination, all of the mice were intravenouslyinjected with 2.5×10⁵ CFU of M. tuberculosis H37Rv. The mice weresacrificed 4 weeks after injection. The protection levels weredetermined by evaluating the numbers of viable M. tuberculosis H37Rvrecovered from the lungs and spleen of both groups of mice (FIG. 4).Both vaccines conferred similar but significant levels of protection, ifcompared with the controls treated with saline solution (p<0.05).Inhibition of M. tuberculosis H37Rv growth was recorded in both thelungs and the spleen, with reductions of approximately 1.5 log₁₀ and 1.3log₁₀ CFU, respectively.

Example 6 Protective Immunity of SO2 of the Present Invention in GuineaPigs

The results obtained in mouse vaccination experiments indicated that theattenuation of the SO2 strain of the present invention gave it vaccineproperties similar to those of BCG Pasteur. However, it is generallyaccepted that guinea pigs are a more appropriate model for humantuberculosis, with many similarities in terms of the progression andpathology of the disease. This animal model is therefore a moreappropriate system for evaluating the efficacy of a vaccine. Toinvestigate the protective efficacy of the SO2 strain of the presentinvention, we carried out experiments that involved aerosol applicationto vaccinated animals at low doses (10-50 CFU) and at high doses (500CFU). Groups of six guinea pigs were subcutaneously vaccinated with SO2of the present invention or with BCG. Ten weeks after vaccination, allthe guinea pigs were administered inhaled doses of M. tuberculosisH37Rv.

The animals that received the lower dose were sacrificed after 4 weeks,and the bacterial loads in the lungs and spleen were counted. Theprotective efficacy was determined by comparing the numbers of viable M.tuberculosis H37Rv recovered from the organs of guinea pigs in eachtreatment group. In this experiment, the reduction of CFUs in the lungsand spleen was significantly different between the unvaccinated controlanimals and those vaccinated with BCG or M. tuberculosis SO2 (p=0.005).However, no significant difference was found between the vaccinatedgroups (FIG. 5).

The guinea pigs that received the high dose were sacrificed 180 daysafter application or when a loss of 20% of the body weight was noted.The protection levels were determined by comparing the survival times ofthe guinea pigs of each treatment group. The progression of developmentof the lesions was also studied in the vaccinated/infected guinea pigsand compared with that observed in the unvaccinated/uninfected animals.During the phase of the experiment subsequent to inhalation, all theunvaccinated guinea pigs and four of the guinea pigs vaccinated with BCGwere sacrificed at the human end point, before the time end point (180days) due to severe and progressive disease (FIG. 6 a). In contrast, allof the guinea pigs vaccinated with the SO2 strain of the presentinvention survived throughout the duration of the study. The guinea pigsvaccinated with the SO2 strain of the present invention survivedsignificantly longer than those vaccinated with BCG (p=0.018), which, inturn, survived significantly longer than the control guinea pigs, whichwere treated with saline solution (p=0.0049). Furthermore, the guineapigs vaccinated with the SO2 strain gained weight and did not presentany visible or clinical sign of disease.

The extent of lung disease, measured by total lung consolidation, alsovaried between the different treatment groups. The highest level ofprogression of the disease was observed, as predicted, in theunvaccinated guinea pigs, and in this group of animals a mean percentageof consolidation of 76% was measured (FIG. 6 b, 6 c). Coalescence ofgranulomas was also pronounced in the guinea pigs vaccinated with BCG,with a mean consolidation of 70% measured in the lungs. In contrast,less consolidation (approximately 50%) was observed in the guinea pigsvaccinated with SO2 of the present invention, this consolidation beingsignificantly less (p<0.05) than with the unvaccinated animals and thosevaccinated with BCG (FIG. 6 c). This reduction in the severity of thedisease was also reflected in the bacterial counts of lung and spleenhomogenates. In the vaccinated groups a difference in the levels ofinhibition of M. tuberculosis H37Rv growth was observed in both organs.The numbers of CFUs recovered from guinea pigs vaccinated with SO2 werereduced by more than 1×log₁₀ compared to those of guinea pigs vaccinatedwith BCG, and this reduction was statistically significant (p<0.05) inthe spleen (FIG. 6 d). These data show that the SO2 strain of thepresent invention was better than BCG at conferring a higher survivalrate on infected guinea pigs, reducing the severity of the disease inthe lungs and preventing the infection from spreading to the spleen.

Example 7 Attenuation of SO2 of the Present Invention is Due to thePhoP− DIM− Double Mutation

Infection studies in Balb/C mice by intravenous injection of the SO2(phoP− DIM−) strain compared to the wild-type MT103 strain and thestrain complemented with phoP (SO2+pSO5) showed that the attenuation ofinfection with SO2 in BalbC mice by intravenous injection is notrestored by complementation with phoP. The reduction of colonies (CFU)in both spleen (FIG. 7 a) and lung (FIG. 7 b), measured after 3 and 6weeks, were not restored in the complemented strain, as it is notvirulent in immunocompetent mice, these experiments suggesting that thesurprising attenuation could be due to a second additional mutation(FIG. 7).

Lipid studies of different strains of M. tuberculosis by thin layerchromatography showed that the SO2 strain does not produce DIM and thisis independent of the phoP mutation (FIG. 8).

To show that SO2 is not toxic, six guinea pigs were inoculated with 50times the vaccine dose. The survival rate was 100% after the 6-monthduration of the experiment. A weight gain was observed in all theanimals over the 6 months, showing the non-toxicity of the SO2 strain(Y=weight in grams and week of infection. X=time in weeks) FIG. 12.

Sensitivity to antituberculosis drugs was also studied. The minimuminhibitory concentration (MIC) was determined for the antituberculosisdrugs Ethambutol, Isoniazid, Rifampicin and Streptomycin against M.tuberculosis strains H37Rv, MT103 (wild type) as the control and the SO2strain. The values (micrograms/ml) indicate that after inactivation ofthe phoP gene the SO2 candidate vaccine strain conserves its sensitivityto the most common drugs clinically used against tuberculosis.

Ethambutol Isoniazid Rifampicin Streptomycin H37Rv 2 0.5 <0.004 <0.5MT103 2 0.5 <0.004 <0.5 SO2 2 0.25 <0.004 <0.5

Studies of attenuation in intratracheally inoculated BalbC mice showedthat with the M. tuberculosis DIM− (1A29) strain 50% of the mice hadsurvived after 20 weeks. All of the animals inoculated with SO2 (phoP−and DIM− mutant) surprisingly survived for the 20 weeks of theexperiment (FIG. 11).

Example 8 Protection of SO2 of the Present Invention is Due to the PhoP−DIM− Double Mutation

Protection was studied in guinea pigs vaccinated and infected by aerosolwith M. tuberculosis H37Rv. Guinea pig survival after 300 days. Aftersubcutaneous vaccination the animals are infected with a virulent strainof M. tuberculosis (H37Rv) at a high dose to study survival. After 60days the 6 guinea pigs that had not been vaccinated had died, whilst thegroups vaccinated with SO2, phoP− and BCG had survived. After 300 daysof infection 3 guinea pigs vaccinated with BCG and phoP− had died,compared to only one of the group vaccinated with SO2, which indicatesthat the protection of the phoP mutant is similar to that of the currentvaccine BCG, whereas vaccination with SO2, the phoP− and DIM− doublemutant, protects better in the guinea pig model (FIG. 13).

These protection studies in guinea pigs lasted 400 days, but the 6unvaccinated guinea pigs had died after 60 days. After 400 days ofinfection 3 guinea pigs from the group vaccinated with SO2 (FIG. 14 a)survived, whereas just 1 guinea pig vaccinated with BCG (FIG. 14 a andFIG. 14 b) and phoP− (FIG. 14 b) had survived, indicating again that theprotection of the phoP mutant is similar to that of BCG, whilstvaccination with SO2, the phoP− and DIM double mutant, protects betterafter the 400 days of the experiment.

Example 9 Construction of the Candidate Tuberculosis Vaccine Based onMutation by Deletion of the fadD26 Gene

The M. tuberculosis strains used for the construction of the mutant bydeletion of the fadD26 (ΔfadD26) gene are SO2, which contains the phoPgene inactivated by insertion of a kanamycin resistance cassette, andthe MT103 clinical strain.

-   1. Construction of the plasmids    -   1.1. Cloning of the fadD26 gene, which is involved in DIM        synthesis. The fadD26 gene was amplified by PCR, using genomic        DNA from M. tuberculosis H37Rv and using primers fadD26Fw (SEQ        ID NO:1) and fadD26Rv (SEQ ID NO:2). The PCR product was        inserted into the pGEM-T Easy vector (Promega) to construct        plasmid pAZ1.    -   1.2. Deletion of the fadD26 gene and insertion of the hygromycin        resistance cassette. A BamHI-EcoRV fragment of pWM27 (Malaga et        al., FEMS Microbiology letters 219 (2003) 261-268), which        contains the res-Ωhyg-res cassette (the res sites, recognised by        γδ resolvase, will make it possible to eliminate the resistance        marker in a second passage), was inserted between the        BamHI-EcoRV sites of fadD26 in pAZ1 to construct pAZ3.    -   1.3. Construction of the suicide vector for inactivation of the        gene by homologous recombination. Plasmid pAZ3 was digested with        XhoI, releasing the fadD26::Ωhyg insert, which was incorporated        into the pJQ200X vector, linearised with the same enzyme. The        final plasmid was named pAZ5.-   2. Construction of the M. tuberculosis DIM− strains    -   2.1. Plasmid pAZ5 was inserted into the M. tuberculosis SO2 and        MT103 strains.    -   2.2. Selection of the single recombinants. Culture in hygromycin        (20 μg/ml) of the bacteria that include the plasmid and checking        for its resistance to gentamicin (10 μg/ml).    -   2.3. Selection of the double recombinants. Culture of the single        recombinants in sucrose 2% (Pelicic et al. 1997) and hygromycin        and checking for their sensitivity to gentamicin.-   3. Elimination of the antibiotic resistance marker from the ΔfadD26    mutation.    -   3.1. To eliminate the res-Ωhyg-res cassette and produce the        mutation without an antibiotic resistance marker, plasmid pWM19,        which contains γδ resolvase, is inserted and selected by        gentamicin resistance. Then the plasmid is eliminated by        incubating at 39° C. in sucrose 2% (Malaga et al. 2003).

Example 2.2

The M. tuberculosis strain used for the construction of the doublemutant by deletion ΔphoP ΔfadD26 is MT103 ΔfadD26.

-   4. Construction of the plasmids    -   4.1. Cloning of the phoP gene. The phoP gene was amplified by        PCR, using genomic DNA from M. tuberculosis H37Rv and using        primers phoPF (SEQ ID NO:3) and phoPR (SEQ ID NO:4). The PCR        product was inserted into the pGEM-T Easy vector (Promega) to        construct plasmid pAZ11.    -   4.2. Deletion of the phoP gene and insertion of the kanamycin        resistance cassette. A BamHI-EcoRV fragment of pCG122 (Malaga et        al. 2003), which contains the res-Ωkm-res cassette, was inserted        between the BclI-EcoRV sites of phoP in pAZ11 to construct        pAZ13.    -   4.3. Construction of the suicide vector for inactivation of the        gene by homologous recombination. Plasmid pAZ13 was digested        with XhoI, releasing the phoP::Ωkm insert, which was        incorporated into the pJQ200X vector, linearised with the same        enzyme. The final plasmid was named pAZ15.-   5. Construction of the M. tuberculosis ΔphoP ΔfadD26 double mutant    strain.    -   5.1. Plasmid pAZ15 will be inserted into the M. tuberculosis        MT103 ΔfadD26 strain.    -   5.2. Selection of the single recombinants. Culture in kanamycin        (20 μg/ml) of the bacteria that include the plasmid and checking        for its resistance to gentamicin (10 μg/ml).    -   5.3. Selection of the double recombinants. Culture of the single        recombinants in sucrose 2% (Pelicic et al. 1997) and kanamycin        and checking for their sensitivity to gentamicin.-   6. Elimination of the antibiotic resistance marker from the ΔphoP    mutation.    -   6.1. To eliminate the res-Ωkm-res cassette and produce the        mutation without an antibiotic resistance marker, plasmid pWM19,        which contains γδ resolvase, will be inserted and selected by        hygromycin resistance (20 μml). Then the plasmid will be        eliminated by incubating at 39° C. in sucrose 2% (Malaga et al.        2003).

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The invention claimed is:
 1. A method of treating bladder cancer in ahuman or animal, said method comprising the step of administering to apatient in need thereof an effective amount of a pharmaceuticalformulation comprising an isolated microorganism belonging to theMycobacterium tuberculosis complex, wherein said isolated microorganismis Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacteriumafricanum, and at least one pharmacologically suitable excipient,wherein in said microorganism (a) the phoP gene is inactivated ordeleted; and (b) a second gene is inactivated or deleted to preventphthiocerol dimycocerosates (“DIM”) production.
 2. A method of treatingbladder cancer in a human or animal according to claim 1 wherein DIMproduction is inactivated through the deletion or inactivation of thefadD26 gene.
 3. A method of treating bladder cancer in a human or animalaccording to claim 2 wherein the isolated microorganism is Mycobacteriumtuberculosis.